1. Field of the Invention
The present invention concerns methodology to be utilized with a probe operative in the mid-infrared or near-infrared region of the electromagnetic spectrum for the sensing of the absorption of infrared energy in a sample for the determination of the ultrastructure of connective tissue, such as soft connective tissue, such as cartilage, either in vivo or in vitro.
2. Background Information
The repair of defects in articular cartilage remains a challenging problem in orthopaedic surgery. Recently, novel tissue engineering technologies have facilitated the synthesis of cartilage-like tissue for potential implantation into defect sites. Commensurate with such developments is the requirement for new methodology to evaluate the integration of these matrices into cartilage and to assess their capability for regeneration and repair of tissue.
Fourier transform infrared (“FT-IR”) spectroscopy has been used extensively to study the structure and orientation of proteins, lipids and inorganic compounds in numerous normal and pathological tissues (for review, see: Infrared and Raman Spectroscopy of Biological Materials, Eds. Gremlich, H. U., Yan, B., New York, Marcel-Dekker, (2001); also see Bychkov, S. M. and Kuzmina, S. A., “Study of Tissue Proteoglycans by Means of Infrared Spectroscopy”, Biull. Eksp. Biol. Med., 114, pp. 246-249, (1992); Camacho, N. P., Hou, L., Toledano, T. R., Ilg, W. A., Brayton, C. F., Raggio, C. L., Root, L., and Boskey, A. L., “The Material Basis for Reduced Mechanical Properties in Oim Mice Bones”, J. Bone Miner. Res., 14, pp. 264-272, (1999); Lazarev, Y. A., Grishkovsky, B. A., and Khromova, T. B., “Amide I Band of IR Spectrum and Structure of Collagen and Related Polypeptides”, Biopolymers, 24, pp. 1449-1478, (1985); Liu, K. Z., Dembinski, T. C., and Mantsch, H. H., “Rapid Determination of Fetal Lung Maturity from Infrared Spectra of Amniotic Fluid”, Am. J. Obstet. Gynecol., 178, pp. 234-241, (1998); Mendelsohn, R., and Moore, D. J., “Vibrational Spectroscopic Studies of Lipid Domains in Biomembranes and Model Systems”, Chem. Phys. Lipids, 96, pp. 141-157, (1998); Moore, D. J., Rerek, M. E., and Mendelsohn, R., “Lipid Domains and Orthorhombic Phases in Model Stratum Corneum: Evidence from Fourier Transform Infrared Spectroscopy Studies”, Biochem. Biophys. Res. Commun., 231, pp. 797-801, (1997); Moore, D. J., Gioioso, S., Sills, R. H., and Mendelsohn, R., “Some Relationships Between Membrane Phospholipid Domains, Conformational Order, and Cell Shape in Intact Human Erythrocytes”, Biochim. Biophys. Acta., 1415, pp. 342-348, (1999)).
The coupling of an FT-IR spectrometer to an optical microscope (FT-IR microspectroscopy (“FT-IRM”)) permits quantitation of the relative amounts, molecular nature, distribution and orientation of these compounds at a spatial resolution of approximately 10 μm. Recent studies have utilized this technique to evaluate change in the mineral and organic phase in normal (Paschalis, E. P., Betts, F., DiCarlo, E., Mendelsohn, R., and Boskey, A. L., “FTIR Microspectroscopic Analysis of Normal Human Cortical and Trabecular Bone”, Calcif, Tissue Int., 61, pp. 480-486, (1997)) and osteoporotic human bone (Paschalis, E. P., Betts, F., DiCarlo, E., Mendelsohn, R., and Boskey, A. L., “FTIR Microspectroscopic Analysis of Human Iliac Crest Biopsies from Untreated Osteoporotic Bone”, Calcif. Tissue Int., 61, pp. 487-492, (1997)), in bones from mouse models of osteogenesis imperfecta (Camacho, N. P., Landis, W. J., and Boskey, A. L., “Mineral Changes in a Mouse Model of Osteogenesis Imperfecta Detected by Fourier Transform Infrared Microscopy”, Connect. Tissue Res., 35, pp. 259-265, (1996) and X-linked hypophosphatemia (Camacho, N. P., Rimnac, C. M., Meyer, R. A. J., Doty, S., and Boskey, A. L., “Effect of Abnormal Mineralization on the Mechanical Behavior of X-Linked Hypophosphatemic Mice Femora [published erratum appears in Bone, 1996 July 19(1);77], Bone, 17, pp. 271-278, (1995)) in turkey tendon (Gadaleta, S. J., Camacho, N. P., Mendelsohn, R., and Boskey, A. L., “Fourier Transform Infrared Microscopy of Calcified Turkey Leg Tendon”, Calcif. Tissue Int., 58, pp. 17-23, (1996)), and in mineralizing chick limb bud cell cultures (Boskey, A. L., Guidon, P., Doty, S. B., Stiner, D., Leboy, P., and Binderman, I., “The Mechanism of Beta-Glycerophosphate Action in Mineralizing Chick Limb-Bud Mesenchymal Cell Cultures”, J. Bone Miner. Res., 11, pp. 1694-1702, (1996).
A powerful enhancement to this technique has been the recent development of an infrared focal plane array detector of the FT-IR microscope. This technology enables 4096 individual spectra to be collected simultaneously over a 400×400 μm2 region at 7 microns spatial resolution in less than 10 minutes; an extraordinary reduction in time and effort compared to conventional FT-IR microscopy. Moreover, infrared images based on the spatial distribution of specific molecular species in biological tissues can now easily be generated (Kidder, L. H., Kalasinsky, V. F., Luke, J. L., Levin, I. W., and Lewis, E. N., “Visualization of Silicone Gel in Human Breast Tissue Using New Infrared Imaging Spectroscopy”, Nat. Med., 3, pp. 235-237, (1997); Lewis, E. N., Kidder, L. H., Levin, I. W., Kalasinsky, V. F., Hanig, J. P., and Lester, D. S., “Applications of Fourier Transform Infrared Imaging Microscopy in Neurotoxicity”, Ann. N.Y. Acad., Sci., 820, pp. 234-247, (1997); Marcott, C., Reeder, R. C., Paschalis, E. P., Tatakis, D. N., Boskey, A. L., and Mendelsohn, R., “Infrared Microspectroscopic Imaging of Biomineralized Tissues Using a Mercury-Cadmium-Telluride Focal-Plane Array Detector”, Cell. Mol. Biol. (Noisy-le-grand), 44, pp. 109-115, (1998)).
FT-IR microscopic determination of collagen orientation in articular cartilage was discussed in Camacho, N. P., Mendelsohn, R., Grigiene, R., Torzilla, P. A., “Polarized FI-IR Microscopic Determination of Collagen Orientation in Articular Cartilage”, 42nd Annual Meeting, Orthopaedic Research Society, Feb. 19-22, 1996, Atlanta, Ga. FT-IR microscopic imaging of the major components of articular cartilage was discussed in “FT-IR Microscopic Imaging of Collagen and Proteoglycan in Bovine Cartilage”, Camacho, N. P.; West, P.; Torzilli, P. A.; Mendelsohn, R., BioPolymers, 62:1-8 (2001). FT-IR microscopic imaging analysis of bovine nasal cartilage components utilizing multivariate analysis was discussed in Potter, K., Kidder, L. H., Levin, I. W., Lewis E. N., Spencer R. G., Arthritis & Rheum, 44(4): 846-855 (2001).
Heretofore, articular cartilage, a connective tissue that provides resistance to compressive forces during joint movements, had not been examined in detail by conventional FT-IR spectroscopy. In its normal state, articular cartilage displays distinct microscopic zonal heterogeneity that is well-suited to FT-IRM analysis. The framework of cartilage is composed of a network of type II collagen fibrils that interact with type IX and XI collagens, non-collagenous proteins and proteoglycan (PG) components (Pelletier, J., and Martel-Pelletier, J., “The Musculoskeletal System: Articular Cartilage”, Schumacher, H. R., Klippel, J. H., and Koopman, W. J., Primer on the Rheumatic Diseases, Atlanta: The Arthritis Foundation, pp. 8-10, (1993)).
The surface layer of tissue (superficial tangential zone) is composed of fibrils oriented parallel to the surface, presumably to minimize leakage of the tissue components (PGs and water) during loading. The midzone (or transitional zone) has been reported to have fibrils perpendicular and parallel to the surface, but also may contain fibrils in a non-specifically oriented network. The deep zone, adjacent to the bone, contains fibrils oriented parallel to the long bone axis, that may serve to strengthen the bone-cartilage junction. In addition to heterogeneity with respect to the orientation of collagen, the concentrations of individual tissue components, such as proteoglycans, vary zonally in normal cartilage.
Although both collagen (Lazarev, Y. A., Grishkovsky, B. A., and Khromova, T. B., “Amide I Band of IR Spectrum and Structure of Collagen and Related Polypeptides”, Biopolymers, 24, pp. 1449-1478, (1985); Fraser, R. D. B. and MacRae, T. P.; Collagen; Horecker, B., Kaplan, N. O., Marmur, J., and Scheraga, H. A., Conformation in Fibrous Proteins and Related Synthetic Polypeptides, New York: Academic Press, pp. 344-402, (1973); George, A., and Veis, A., “FTIRS in H2O Demonstrates that Collagen Monomers Undergo a Conformational Transition Prior to Thermal Self-Assembly In Vitro”, Biochemistry, 30, pp. 2372-2377, (1991); Lazarev, Y. A., Grishkovsky, B. A., Khromova, T. B., Lazareva, A. V., and Grechishko, V. S., “Bound Water in Collagen-Like Triple Helical Structure”, Biopolymers, 32, pp. 189-195, (1992)) and proteoglycans (Bychkov, S. M., and Kuzmina, S. A., “Study of Tissue Proteoglycans by Means of Infrared Spectroscopy”, Biull. Eksp. Biol. Med., 114, pp. 246-249 (1992); Bychkov, S. M., Bogatov, V. N., and Kuzmina, S. A., “Infrared Spectra of Cartilage Proteoglycans”, Bull. Eksp. Biol. Med., 90, pp. 561-563, (1980); Bychkov, S. M., Bogatov, V. N., and Kuzmina, S. A., “Study of Different Proteoglycan Salts”, Bull. Eksp. Biol. Med., 92, pp. 302-305, (1981)) have been examined individually by infrared spectroscopy, but prior to the present invention, they had not been examined by IR in the intact cartilagineous tissues.
U.S. Pat. No. 5,170,056 to Berard et al. (the entire contents of which are incorporated by reference herein) concerns a probe operative in the infrared region of the electromagnetic spectrum in situ sensing of the absorption of IR energy in a sample.
U.S. Pat. No. 5,280,788 discloses an optical needle device for the diagnosis of tissues, but cartilage is not discussed therein.
U.S. Pat. No. 5,923,808 to Melling (the entire contents of which are incorporated by reference herein) describes a mid-infrared spectroscopic probe attached to a fiber-optic cable.
U.S. Pat. Nos. 5,701,913 and 6,068,604 concern probes for measuring the stiffness of cartilage or cartilage compressive properties by disposing a probe against a tissue, applying force and measuring the response to the force (the relative displacement of the probe). U.S. Pat. Nos. 5,701,913 and 6,068,604 do not involve the measurement of cartilage properties by radiation.
U.S. Pat. Nos. 5,460,182; 5,769,791; 5,785,658; 5,762,609; 5,772,597; 5,807,261 and 5,987,346 are directed to tissue penetrating devices and sensors for in vivo measurements of body tissues. Re. 36,044 concerns a diagnostic monitor for classifying an unknown biological tissue into two or more types.